CHARACTERIZATION OF THE PREOPERATIVE IMMUNE PROFILE IN A COHORT OF PATIENTS UNDERGOING CARDIOPULMONARY BYPASS SURGERY TO PREDICT POSTOPERATIVE ANTIBODY PRODUCTION AGAINST PF4/H COMPLEXES

Abstract

Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by a lowered platelet count (50% from baseline) 4-10 days after heparin exposure. Autoantibodies specific for platelet factor 4 (PF4) bind PF4 and heparin complexes (PF4/H) and activate platelets through the FcgammaRIIA receptor. Severe cases of HIT can result in thrombotic complications including deep vein thrombosis, pulmonary embolism, and death. Pathogenic class-switched antibodies against PF4/H (IgG) are detectable in circulation as early as five days post-heparin exposure and peak at 14 days. The timeline and class of antibody found in HIT patients suggest that there must be pre-existing immunity against PF4/H. Thus, B cells producing anti-PF4/H antibodies must exist prior to heparin exposure. Cardiac surgery patients are disproportionately prone to anti-PF4/H seroconversion (up to 70%) and thus are utilized in this study as a model patient group. Research objective: The objective of this study is to determine whether the preoperative immune profile is associated with postoperative anti-PF4/H antibody production in a cohort of patients undergoing cardiac pulmonary bypass (CPB) surgery. Materials and methods: To characterize the preoperative immune profile, we used 1) a peripheral blood mononuclear cell (PBMC) enzyme linked immunospot (ELISPOT) assay to measure the prevalence of preoperative anti-PF4/H specific antibody secreting cells (ASC) and 2) a PF4/H-dependant enzyme immunoassay (EIA) to measure the anti-PF4/H antibodies produced by PBMCs in vitro. To characterize postoperative anti-PF4/H seroconversion in CPB patients, we used a PF4/H dependent EIA to measure in vivo levels of anti-PF4/H antibodies produced postoperatively. We also utilize a functional assay, 14C-serotonin release assay (SRA) to determine if seroconverting patients produced platelet activating antibody. Results: All patients were able to produce anti-PF4/H spots in the ELISPOT; however, this did not correlate with the titer of antibody production in vitro nor did it correlate with antibody production in the postoperative period. Instead, we found that pre-operative in vitro anti-PF4/H IgM production was associated with post-operative IgG anti-PF4/H seroconversion (Spearman’s r=0.39, P=0.018). We observed that 92.1% of CPB patients produced PF4/H antibody at postoperative week 3 with some combination of IgA, IgG, and IgM. Of the anti-PF4/H seropositive patients, 26% developed platelet activating antibody and were found seropositive when the SRA was supplemented with PF4 instead of heparin, while 15.7% were seropositive in the original SRA. It was noted that 4 of 10 patients that caused the most robust platelet activation were also seropositive for anti-PF4/H IgA antibody. Lastly, throughout this serosurveillance study, several patients that demonstrated unique immunological features are presented in this study as case studies. Specifically, we report the preoperative, surgical, clinical and postoperative characteristics for 3 patients of interest: 1) in a preoperative setting, a CPB patient’s PBMC were able to be activated and produce anti-PF4/H IgG antibody in vitro, 2) the second patient had platelet-activating antibodies in circulation prior to intraoperative heparin challenge and early post-surgery 3) the third patient who developed probable HIT. Conclusions: Based on our findings, we conclude that preoperative PF4/H ELISPOTs were unable to predict post-operative production of anti PF4/H antibodies. However, preoperative in vitro production of anti-PF4/H IgM may be associated with postoperative production of anti-PF4/IgG antibody and should be investigated further as this may help to elucidate the mechanisms for anti-PF4/H production related to HIT.